“…since stem cells are now being more routinely used for regenerative medicine such as repair of severed spinal cord (Lu et al. 2012), it behooves us to better learn the molecular mechanisms that keeps these invaluable cells in an undifferentiated state so that we can harness their full therapeutic potential.”

Discovery of Primary Cilia in Stem Cells

The primary cilium is organelle that has garnered much attention in the field of cell biology during the last 15 years. It is a slender, solitary hair-like organelle that extends 5-10 uM from each mammalian cell (in the G0 cell cycle state) that is microtubule-based (9 outer doublets arranged in a circular fashion) and dependent on a process called Intraflagellar Transport (IFT). IFT is the bidirectional movement of motors (kinesin-2 in the anterograde and dynein-2 in the retrograde direction) responsible for the assembly and maintenance of the cilium (Pedersen et al., 2006).

Until this time, it had been labeled a ‘vestigial’ organelle not worthy of research. Yet, a breakthrough into the sensory role of the primary cilium came in 2000 based on Dr. Rosenbaum’s research on Chlamydomonas and the motile cilium or flagella. Along with Dr. George Whitman’s group, they were able to show the importance of Tg737 (IFT88) protein to the pathology of polycystic kidney disease in mouse (Pazour et al., 2000). Since then, research into the primary cilium has exploded and has been linked to diverse pathologies (collectively known as ciliopathies) such as

signal transduction pathways have been found to be coordinated by the primary cilia such as hedgehog, wnt, PDGF among others (Veland et al., 2008).

Thus, in 2006, the Christensen lab at the University of Copenhagen (Denmark) with the collaboration of Dr. Peter Satir’s group at Albert Einstein College of Medicine (Bronx, NY) began to investigate whether the human embryonic stem cells (hESCs) possess primary cilium and then to begin preliminary molecular dissections of the role that this organelle could play in the proliferation and differentiation profiles of these pluripotent cells. The Albert Einstein group, due to NIH restrictions, had to work with two federally-sanctioned cell lines. Working with the Laboratory of Reproductive Biology at RigsHospital, the Danish side had access to in-house derived stem cell lines from discarded blastocysts. The advantage for the Danish side was obvious since these newer cell lines hadn’t undergone as many passages as the NIH cell lines and were thus more robust. To begin preliminary characterizations of these lines, some basic hallmarks of hESCs (Bernhardt et al., 2012) had to be localized to the nucleus such as the transcription factor (TF) Oct4 (Fig. 1).

In addition, a single primary cilium can be seen denoted by the acetylated tubulin staining emanating from each cell in the micrographs. Also, the base of the cilium is marked by the presence of pericentrin and centrin which demarcate the centriole.

Together, the three labs were the first to discover primary cilia in stem cells while other groups have since then confirmed these findings(Kiprilov et al. 2008; Han et al. 2008). Attention was then to characterize different signal transduction pathways in the stem cell cilium.Since the hedgehog pathway has been shown to be important for differentiation and proliferation (Cerdan and Bhatia, 2012), the groups characterized this signal pathway in these cells using immunofluorescence, electron microscopy and qPCR techniques. One particularly interesting experiment to show that the hedgehog pathway was functional in these cells was to add the hedgehog agonist, SAG (Smoothened agonist), and then to isolate the cells for immunofluorescence at different times.

Gradually, one can see the appearance of the smoothened protein into the cilium as indicated by increasing intensity of the immunofluorescence staining. Conversely, patched levels in the cilium, decreased. This is a hallmark of hedgehog activation (Fig. 2).Fig. 2 Immunofluorescence micrographs of hESC showing smoothened (green), acetylated tubulin (red) and DAPI (blue). The micrographs from left to right represents SAG treatments at t = 0, 1 and 4 hours.

However, an additional interesting observation was made concerning these stem cells. An important characteristic for stem cells is the presence of certain transcription factors which render these cells in the pluripotent or undifferentiated state. These include Oct4, Sox2, and Nanog whose localization had been observed in the nucleus as expected for other TFs.

However, the Danish groups curiously found a subpopulation of stem cells where these TFs were additionally localized to the primary cilium (Fig. 3). This had never been observed or investigated before. Additionally, proper negative controls were carried out to exclude this phenomenon from being an artifact (e.g. bleed through). Fig. 3 Stem cell markers (Sox2, Nanog, and Oct4) localizing to the nucleus and the primary cilia (arrows) of hESC line LRB003. This and the previous figure show shifted overlay images whereby the green and red channels have been slightly shifted so that the red channel doesn’t swamp out the intensity of the green channels.

Thus, it raises an intriguing possibility that perhaps the primary cilia plays a previously uncharacterized role in the differentiation/proliferation state of the hESCs via possible modifications of these TFs perhaps analogous to the processing of the Gli transcription factors (Hui and Angers, 2011). Another curious observation is that the subpopulation of cells whose primary cilia are positive for these TFs always occur in clusters which might hint at its mechanistic explanation. In conclusion, since stem cells are now being more routinely used for regenerative medicine such as repair of severed spinal cord (Lu et al. 2012), it behooves us to better learn the molecular mechanisms that keeps these invaluable cells in an undifferentiated state so that we can harness their full therapeutic potential.

“…findings from this study demonstrate the feasibility and safety of Stem Cell Educator therapy and demonstrate that Type 1 Diabetes patients achieve improved metabolic control and reduced autoimmunity that lasts months following a single treatment.” “…clinical data provide powerful evidence that reversal of autoimmunity leads to regeneration of islet β cells and improvement of metabolic control in long-standing Type 1 Diabetes subjects. This principle may also be beneficial in the treatment of other autoimmune-related diseases.”

Type 1 diabetes is caused by the body’s own immune system attacking its pancreatic islet beta cells and requires daily injections of insulin to regulate the patient’s blood glucose levels. A new method described in BioMed Central‘s open access journal BMC Medicine uses stem cells from cord blood to re-educate a diabetic’s own T cells and consequently restart pancreatic function reducing the need for insulin.

Stem Cell Educator therapy slowly passes lymphocytes separated from a patient’s blood over immobilized cord blood stem cells (CBSC) from healthy donors. After two to three hours in the device the re-educated lymphocytes are returned to the patient. The progress of the patients was checked at 4, 12, 24 and 40 weeks after therapy.

C-peptide is a protein fragment made as a by-product of insulin manufacture and can be used to determine how well beta cells are working. By 12 weeks after treatment all the patients who received the therapy had improved levels of C-peptide. This continued to improve at 24 weeks and was maintained to the end of the study. This meant that the daily dose of insulin required to maintain their blood glucose levels could be reduced. In accordance with these results the glycated hemoglobin (HbA1C) indicator of long term glucose control also dropped for people receiving the treatment, but not the control group.

Dr Yong Zhao, from University of Illinois at Chicago, who led the multi-centre research, explained: “We also saw an improved autoimmune control in these patients. Stem Cell Educator therapy increased the percentage of regulatory T lymphocytes in the blood of people in the treatment group. Other markers of immune function, such as TGF-beta1 also improved. Our results suggest that it is this improvement in autoimmune control, mediated by the autoimmune regulator AIRE in the CBSC, which allows the pancreatic islet beta cells to recover.”